The present invention is directed to nucleic acid sequences encoding a novel regulatory element that induces a high level of expression to operably linked genes, upon exposure to anaerobic conditions.
Pyruvate decarboxylase (PDC) is a critical enzyme in the anaerobic-specific fermentation pathway. PDC nonoxidatively decarboxylates pyruvate to acetaldehyde which is very toxic to plants. Acetaldehyde is then reduced to ethanol by alcohol dehydrogenase (ADH), regenerating NAD+ which is then utilized in the glycolytic pathway to maintain carbon flow through this pathway under anaerobic conditions. Switching energy production from aerobic glycolysis to anaerobic fermentation is one of the major metabolic adaptations plants undertake when they are submerged or confronted with a lack of oxygen. The importance of increased rates of alcoholic fermentation (AF) under anaerobic conditions was demonstrated by several experimental observations:
(i) enzymes for AF often increase;
(ii) mutants without ADH die more rapidly during anoxia;
(iii) increased tolerance to anoxia comes from hypoxic pretreatments and presumably induction of enzymes of AF;
(iv) high sugar supply increases survivalxe2x80x94presumably due to increased rates of AF; and
(v) rates of AF are related to tolerance of several species to waterlogging or flooding. It has also been suggested that the rate of AF is limited by PDC.
The role of ADH in metabolism and survival of anoxic maize root tips has been investigated by comparing the ethanol production of isogenic lines differing in ADH activity over a xcx9c200-fold range. It was concluded that ADH activity in wild-type maize root tips was not a limiting factor for energy production via fermentation and did not determine viability under anoxia. This conclusion was further supported by additional experiments showing that 70% of the hypoxia acclimated root tips of Adhl null maize survived up to 24 hours of anoxia, whereas only 10% of the unacclimated root tips survived for 6 hours of anoxia. It was also concluded that the high levels of ADH activity inducible in acclimated wild-type maize root tips are in excess of that required to increase rates of fermentation. Thus, PDC, being the first enzyme in the AF pathway, may play a key regulatory role in energy production in cells exposed to hypoxic conditions.
It has been reported that over-expression of a pdc gene from Zymonionas mobilis in tobacco cells results in higher levels of acetaldehyde and ethanol formation, supporting the idea that PDC is likely to be the key regulator of anaerobic metabolism. Unfortunately, the measurements made in this study were only up to 24 hours after anoxia treatment which did not allow for an evaluation of tolerance under long-term anoxia. Moreover, rice might have different mechanisms of submergence tolerance than tobacco as evidenced by the fact that it is relatively more tolerant among other monocots.
Genes encoding PDC have been cloned and characterized from maize (Kelley et al., Plant Mol. Biol. 17: 1259-1261, 1991), yeast (Kellerman et al., Nucl. Acids Res. 14: 8963-8977, 1986), and bacteria (Conway et al., J. Bact. 169: 949-954, 1987). Recently, the isolation and characterization of two pdc cDNA""s and two genomic clones from rice have been reported, and an additional partial cDNA clone named pdc4 has also been reported. The present invention is directed to the characterization of the pdc2 gene and the relative induction of the pdc1 and pdc2 genes over time in both shoots and roots under anaerobic conditions. The present disclosure also presents the map locations of these genes on rice chromosomes and predicts the locations of orthologous loci on maize, oat, and Triticeae chromosomes. The evolutionary relationships among pdc genes from rice, maize, yeast, and bacteria are also discussed.